Composition and method for the treatment of sickle cell anemia

ABSTRACT

The major component of the carbonate fraction of free acids extractable from alfalfa has shown excellent control of symptoms in patients with sickle cell disease. Its structure has been established by proton NMR spectrometry as beign 16-hydroxy-9Z,12Z,14E-octadecatrienoic acid and closely related compounds, such as simple esters, amides, triglycerides, or other derivatives of the carboxylic acid function, and a method for its synthesis from linseed oil or methyl linolenate has been developed. The product from linseed oil, both in the form of the initially formed triglyceride and in the form of its free acid obtained by saponification, is useful for the treatment of sickle cell disease.

CROSS REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part application of copendingapplication Ser. No. 174,602, of Sunday O. Fadulu and Alfred J.Weinheimer, filed on Mar. 20, 1988, now abandoned which is in turn acontinuation-in-part application of application Ser. No. 041,035, ofSunday O. Fadulu and Alfred J. Weinheimer, filed on Apr. 22, 1987, nowabandoned.

BACKGROUND OF THE INVENTION

The present invention relates to 16-hydroxy-9Z,12Z,14E-octadecatrienoicacid and closely related compounds, such as simple esters, amides,triglycerides, or other derivatives of the carboxylic acid function;isolated from the carbonate fraction of free acids extractable fromvarious plant materials, particularly alfalfa. The invention alsorelates to compositions, containing the compounds, which are used forretarding red blood cell sickling associated with sickle cell disease.

Sickle cell disease is an inherited disease stemming from inadequateoxygen transport by an abnormal type of hemoglobin molecule in the redblood cells. It is an inherited disease which can be passed to offspringonly if both parents carry the genetic trait. The trait carriers show nosign of the disease, but statistically one in four of their childrenwill be afflicted with the disease. The disease is most prevalent in theblack races, but is also known in other races surrounding theMediterranean Sea and in India. It affects about 0.2% of the U.S. blackpopulation but is much more prevalent in central Africa.

The most common manifestation of the disease is an extremely painful"crisis," typically lasting several days, and affecting one or anotherlocal part of the body. The crisis often occurs following physicalstress, and appears to be due to limited oxygen supply to the affectedpart. This is due to the inferior oxygen-carrying capability of themutant hemoglobin, as well as to its tendency to aggregate in insolublegels within the red blood cell, often leading to a form resembling asickle. The distorted cells no longer freely traverse capillaries,further limiting oxygen supply to the tissues.

Despite the fact that the cause of sickle disease, i.e., the very minorstructural variation in the mutant hemoglobin, has been known for manyyears, little progress has been made in suitable treatment of thedisease. At the present time, the major treatment for the painful crisesis medication for relief of pain, which merely treats the immediatesymptom. Tissue damage, often involving major organs, occurs with eachsuccessive episode of oxygen deprivation, and the cumulative effects ofthe disease are debilitating and life shortening. Those afflicted withsevere forms of the disease usually do not live through teen years.

Based on current knowledge of the disease, it appears feasible todevelop a drug which will alleviate all of the symptoms of the diseaseand provide perfectly normal lives and life-expectancies for patients.This drug would not cure the disease, because it is genetic in origin,but if available, should effectively treat the disease by alleviation orprevention of its symptoms. The requisite capabilities of a potentiallyuseful drug have been defined by the Sickle Cell Disease Branch of theU.S. National Institutes of Health (NIH). The requirements are specifiedin terms of several laboratory bioassays in which a candidate drug mustperform successfully. No drug meeting these requirements has yet beenannounced. The drug of the present invention, however, displayedexcellent responses in all the bioassays, as can be seen in the Examplesbelow.

Fadulu (Fadulu, "Ethyl-Alcohol Extract From Fagara Zanthoxyloides Root:In vitro Effect on Red Blood Cells," Faculty Research Journal, TexasSouthern University, 1:20-31 (1977)) reported in the 1970's that theextract of the African chewing stick, prepared from the roots of thetree Fagara zanthoxyloides, possessed anti-sickling properties in invitro studies.

Chemical studies were initiated to isolate and identify the activeprinciple(s) in the extract responsible for this activity. Systematicfractionation of the extract, coupled with bioassay of the fractionsproduced at each stage, led to the isolation of a small amount of amixture of rather polar compounds.

This mixture showed good activity in the blood-agar plate test developedby Fadulu to test for anti-sickling drugs. Sheep blood is dispersed inagar in a standard agar plate. On heating in a laboratory oven at 70° C.for 15 minutes, the red plates turn rust brown. If a drop of a solutionof an effective anti-sickling drug is placed on the plate prior toheating, the blood under that spot remains red while the rest of theplate turns brown. The ready availability of this simple test sped theprogress of the chemical work since fractions could be evaluatedimmediately by the chemical workers.

The chromatographic behavior of the components in the active fractionstrongly suggested that they were acidic. Specifically, their TLC spotstailed badly, but were improved by the addition of a trace of aceticacid to the eluting solution. That they were indeed acids was confirmedby dissolving them in base, and then precipitating them with acid. Theirpresence as the free acids in the Fagara extract was confirmed in asimilar experiment which resulted in their direct extraction, along withother acids, from the extract.

Because Fagara root was extremely difficult to obtain in adequatequantities, a quick survey of other plant materials was undertaken withthe objective of locating a more readily available source. It wassomewhat surprising to find that each of the new plant materialsevaluated, primarily green vegetables, afforded the same TLC group ofpolar acids when extracted with dilute base. Additionally, each of thecrude extracts showed good anti-sickling activity on red blood cells invitro using the blood-agar plate test described above. Included werespinach, species spinacia oleracea, mustard greens, species Brassicajuncea, grass clippings, species Stenotaphrum secundatum, alfalfa,species Medicago sativa, and even fallen oak leaves, species Quercusnigra. The greatest quantities of acids were found in alfalfa, nextgreatest in hay, followed by Fagara, the reference point. Lesserquantities were present in grass clippings, still less in spinach andmustard greens, and least in oak leaves. Further, alfalfa, then hay,contained greater proportions of the most polar components. The lesspolar acids were common stearic, linolenic, linloleic, and oleic acids,which were of no interest. The alfalfa extract contained the novelcompounds both in greater quantity and proportion; additionally, becausealfalfa is commercially available year-round, alfalfa was selected forfurther study.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide16-hydroxy-9Z,12Z,14E-octadecatrienoic acid and closely relatedcompounds, in the form of the free acids, and in their combined formssuch as simple esters, amides, triglycerides, or other derivatives ofthe carboxylic acid function.

It is a further object to provide a method of making16-hydroxy-9Z,12Z,14E-octadecatrienoic acid and closely relatedcompounds, in the form of the free acids, and in their combined formssuch as simple esters, amides triglycerides, or other derivatives of thecarboxylic acid function from linseed oil or linolenic acid or itsesters or other suitable derivatives.

It is another object of the present invention to provide a compositionwhich is effective in the alleviation of the symptoms of sickle celldisease.

SUMMARY OF THE INVENTION

The mixture of free acids which is extractable from various plantmaterials has been shown to be useful in the treatment of sickle celldisease. It has been found that the mixture of acids can be separatedinto groups which are extractable from ethyl acetate solution by sodiumbicarbonate, or by sodium carbonate, or by neither. The carbonatesoluble fraction of acids showed the best in vitro antisicklingactivity, and was chosen for further study with the objective ofisolating and identifying the active principle(s).

The major component in the mixture proved to be16-hydroxy-9Z,12Z,14E-octadecatrienoic acid (Compound I). Compound I andother closely related compounds, such as its methyl ester andtriglycerides containing Compound I, were synthesized from methyllinolenate and linseed oil.

Compound I and other closely related compounds, such as the methyl esterand triglycerides containing Compound I, were used in compositions whichwere found to be effective in the alleviation of the symptoms of sicklecell disease. The compounds may be mixed with appropriatepharmaceutically acceptable vehicles and administered orally,intravenously, subcutaneously, intraperitoneally, or via suppositories.

DETAILED DESCRIPTION OF THE INVENTION

Commercial alfalfa is available in a finely divided dried form in bags,and in the familiar field-dried bales. The former were used in benchstudies, the latter in pilot scale work. The desired acid fraction wasobtained by soaking the alfalfa in 3% aqueous sodium hydroxide for aminimum of 2 hours followed by filtration, acidification, and extractionof the free acids by hexane. In addition to sodium hydroxide, ammoniumhydroxide and other alkali metal hydroxides such as potassium hydroxidemay be used instead. Although hexane was the solvent of choice becauseit selectively dissolved the active acids, leaving other acidic materialbehind, its use was accompanied by severe emulsion problems. A usefulalternative, particularly in large scale preparations, employed1,1,1-trichloroethane (TCE) as the solvent for segregating the freeacids following acidification of the extract. Other extraction solventssuch as chloroform or methylene chloride may be used in place of TCE.Since considerable quantities of inactive acidic material were presentin the TCE extract, the TCE soluble fraction was subsequentlypartitioned between hexane and methanol containing aqueous acid. Theportion of the acids which thus partitioned into hexane were identicalby thin layer chromatography (TLC) to the acid fraction obtained bydirect extraction with hexane.

The brown-green mixture of acids produced in this fashion was referredto as the hexane fraction. It displayed good anti-sickling activity inthe blood-agar plate test. Purification of the mixture was hindered bythe presence of significant quantities of dark pigments whichco-chromatographed with the desired acids in all systems evaluated.

It was found that the pigment could be overcome by partitioning an ethylacetate solution of the hexane acids into three fractions which were 1)extractable by 5% sodium bicarbonate, 2) extractable by 5% sodiumcarbonate, and 3) extractable by neither. Most of the interferingpigments were found in the bicarbonate fraction, whereas the carbonatefraction was mostly pigment free. The yellow carbonate fraction wasstrongly active in the blood-agar plate test and by TLC showed apredominance of the characteristic spots always associated with activematerial. The bicarbonate fraction was less active, and showed thepresence of some of the desired components, but the TLC pattern wasstrongly overlaid by a continuous streak of pigments. The fractionsoluble in neither bicarbonate or carbonate was not significantlyactive.

Repeated reverse phase chromatography of the free acids in the carbonatefraction on C₁₈ -silica gel was effective in removing the residualpigments, but failed to provide pure compounds as judged by TLC andproton magnetic-resonance (PMR) spectra. However, pure individualcompounds were successfully obtained by chromatography of the mixture ofmethyl esters prepared by treating the enriched fractions of acids fromthe preceding chromatographies with diazomethane. In this manner, onemajor compound, and much smaller proportions of several closely relatedcompounds were isolated in pure or nearly pure form, all as methylesters of the naturally occurring acids. Their molecular weights wereestablished by mass spectrometry and their structures were deduced byPMR spectroscopy. For the major compound, decoupling experiments clearlydelineated all coupling patterns, and thus connectivities, in themolecule.

The major component in the mixture proved to be the methyl ester of16-hydroxy-9Z,12Z,14E-octadecatrienoic acid (Compound II). Its molecularion M⁺ =308, corresponded to the composition C₁₉ H₃₂ O₃. The PMRspectrum showed signals in the low field for a secondary alcohol and sixolefinic protons. The high field region contained signals typical of anunsaturated fatty acid ester. The precise structure of Compound II wasestablished by a series of decoupling experiments. In brief, thedecoupling work showed first that the alcohol function was located atposition 16, since its carbinyl hydrogen showed coupling to a methylenegroup (17) that in turn was coupled to the methyl group (18) at the endof the carbon chain. Proceeding next in the other direction along thechain, the carbinyl hydrogen was shown also to be coupled to a vinylhydrogen (15) which in turn was coupled to another vinyl hydrogen (14),and that to another (13), and that to still another (12). This lastvinyl hydrogen was coupled to a methylene group (11) which, from itschemical shift, was doubly allylic. This required that the remainingunsaturation be located between positions 10 and 9. The signals for H-9,although overlapped with those of H-10 and H-12, showed coupling to anallylic methylene (8).

The methylene (2) in the alpha position to the ester function wasassignable from its chemical shift. This signal was shown to be coupledto another methylene (3) assignable to the β-position. The remainingatoms which were not accounted for in the decoupling experimentsrepresented four methylene groups, which necessarily were situatedbetween atoms 3 and 8, and which complete the structure assignment ofCompound I.

The geometry of the double bonds in the conjugated diene system wereassigned as 12-Z and 14-E (12-cis and 14-trans) on the basis of themagnitudes of their respective vicinal coupling constants. The couplingconstants observed, J₁₂,13 =10.8 Hz and J₁₄,15 =15.2 Hz are typical ofcis and trans double bonds, respectively.

The geometry of the non-conjugated double bond at position 9 could notbe determined from the PMR spectrum since the coupling constant, J₉,10,could not be ascertained. The resonances for the H-9 and H-10 protonswere overlapped with each other and with signal for H-12, and appearedas a non-first-order multiplet. However, as shown below, the geometry ofthis double bond was established as cis because of the identity of theester prepared synthetically from all-cis methyl linolenate acid withthat prepared from the extract.

No attempt was made to determine whether the natural acid or the derivedmethyl ester was optically active. Since the compound was obtained fromnatural sources and may be the result of enzymatic synthesis, it ispossible that the compound occurs in nature as one of the twoenantiomers due to optical activity at the single chiral center,position 16, in the molecule.

Synthesis of Compounds I and II

As a synthetic objective, the structure of Compound I imposes somechallenging and demanding requirements. These center not only on theregiochemical considerations for incorporation of the alcohol functionand three olefinic centers in the proper positions within the eighteencarbon acid skeleton, but also on the more demanding geometricrequirements for each of the double bonds.

Although it is possible to design synthetic approaches by which thedetailed structure of Compound I could be constructed by sequentialelaboration from simpler starting materials, any total syntheticapproach would be both time-consuming and very expensive. The objectivefor a synthesis of Compound I, in addition to confirming the structureassigned by spectral methods, was to prepare Compound I by aninexpensive route, that would make the compound available for use as atherapeutic agent at a reasonable cost. From this practical standpoint,there would in fact be no need that the synthetic drug be of highpurity. A product containing Compound I in even small amounts, rangingfor example from about 0.001 to about 40% of the pharmaceuticalcomposition, should serve quite adequately for clinical use as atherapeutic agent for sickle cell disease, much as the crude extractpreparation, which contained possibly 5% of Compound I, has alreadyshown efficacy in several small clinical trials.

The triply unsaturated C₁₈ fatty acid, linolenic acid, and itsderivatives, such as methyl linolenate, were considered to be apotential starting material for synthesis of Compound I. It is presentin large amounts in the form of triglycerides in certain vegetable oils.One of these, linseed oil, is a readily available and inexpensivearticle of commerce.

The synthesis of Compound I would involve isomerization of the15,16-epoxide of linolenic acid to the allylic alcohol, as follows:##STR1##

This type of transformation is normally effected by use of strong basessuch as lithium diethylamide. Because the methylene group at position 14is allylic to the 12,13-double bond in linolenic acid, its greaterlability (compared to the alternate 17-methylene) would favor thedesired regiochemical course of reaction.

The epoxidation was conducted using equimolar quantities of methyllinolenate and peracid which would be expected to form roughly equalamounts of the three possible epoxide and to avoid or minimize formationof bis-epoxides which could not lead to the desired product. SeeExample 1. The epoxidation mixture, without any purification, wasisomerized by heating with TsOH in benzene for several hours. SeeExample 2. Chromatography of the reaction mixture permitted isolation ofa small amount of Compound II. Its PMR spectrum was identical to that ofCompound II, which had been isolated previously as the major componentof the mixture of methyl esters prepared from the acids of the carbonatefraction from the alfalfa extract, and its COSY spectrum fullysubstantiated the structure assigned earlier to Compound II on the basisof decoupling experiments. Also formed in this reaction was a mixture ofthe 1,2-diol monotosylates resulting from the well-known ring openingreaction of TsOH with epoxides.

In order to produce a large quantity of a form of Compound I for aclinical trail, the reaction sequence was applied next to linseed oil.The natural oil, known as raw linseed oil, is a triglyceride mixture inwhich approximately 50% of the combined acid is linolenic acid. Smallerquantities of linoleic (20%), oleic (20%), and saturated acids (10%) arealso typically present. Epoxidation of raw linseed oil was performedwith an equimolar amount of peracid, followed by TsOH catalyzedisomerization. See Examples 3 and 4. Following normal workup, butwithout further purification, the product of this reaction was used in aclinical trial. A portion of the triglyceride product was saponified toliberate the free acids which were also evaluated in that trial. SeeExample 5. A placebo consisting of raw linseed oil was included. Boththe triglyceride and free acid forms of the drug were highly effectivein eliminating crises and other adverse symptoms in sickle cellpatients. No such effect was observed with the placebo. See Example 6.

EXAMPLE 1 Partial Epoxidation of Methyl Linolenate

A solution of 1.47 g (6.8 mmol) of m-chloroperoxybenzoic acid (80%purity) in 300 ml of methylene chloride was added over a period of threehours to a stirred solution of 5.0 g (17.1 mmol) of methyl linolenate in250 ml of methylene chloride containing 1.4 g (16.6 mmol) of solidsodium bicarbonate. Stirring was continued for one hour after theaddition was complete. A solution of 1.0 g (7.9 mmol) of sodium sulfitein 100 ml of water was then added with continued stirring for another 30minutes. The aqueous layer was separated, washed three times with 50 mlof methylene chloride and the combined organic layers were washed twicewith 50 ml of 5% sodium carbonate, twice with 50 ml of water, and thendried over anhydrous sodium sulfate. Removal of the solvent using arotary evaporator at 40° C. and water aspirator vacuum afforded 4.5 g ofpale yellow oil.

Chromatography of the partially epoxidized methyl linolenate on a silicagel column using a gradient of ethyl acetate in hexane for elutionafforded unchanged methyl linolenate, its 12,13-epoxide, and anunseparated mixture of its 9,10- and 15,16-epoxides. These epoxides werecharacterized by proton NMR. In addition a small amount of slower movingmaterial, probably the diepoxide and possibly triepoxide, was obtainedbut not characterized.

EXAMPLE 2 Isomerization of Methyl Linolenate Epoxides byp-Tolunesulfonic Acid

p-Toluenesulfonic acid (4.0 mg) was added a solution of 4.0 g of thecrude epoxidation product prepared above in 250 ml of dry benzene. Themixture was heated at reflux for 4 hours. The benzene was removed atreduced pressure and the residue was taken up in 200 ml of ethylacetate. The solution was washed with 50 ml of 5% sodium bicarbonate, 50ml of water, and dried over sodium sulfate. Removal of the solvent underreduced pressure afforded a residue of 3.5 g of yellow oil.Chromatography on a silica gel column using a gradient of ethyl acetatein hexane for elution provided a pure sample of methyl16-hydroxy-9Z,12Z,14E-octadecatrienoate. Its proton NMR spectrum wasidentical in all respects with that of the ester of the major componentisolated from the carbonate fraction of the alfalfa extract.

EXAMPLE 3 Partial Epoxidation of Linseed Oil

A solution of m-chloroperoxybenzoic acid (134.0 g, 0.62 mol) in 1 l ofmethylene chloride was added over a period of 2.5 hours to a stirredsolution of 600 g (0.67 mol) of commercial raw linseed oil in 2 l ofmethylene chloride containing 60.0 g (0.71 mol) of sodium bicarbonate atroom temperature. Stirring was continued for one hour after the additionwas complete. A solution of sodium sulfite (50.0 g, 0.4 mol) in 1000 mlof water was then added, with continued stirring for another 30 minutes.The aqueous layer was separated and washed with 300 ml of methylenechloride. The combined organic layers were washed with three 1 lquantities of 5% sodium carbonate, 1 l of water, and dried overanhydrous sodium sulfate. Removal of the solvent under reduced pressureprovided 550 g of a yellow oil.

EXAMPLE 4 Isomerization of Epoxidized Linseed Oil by p-TolunesulfonicAcid

p-Toluenesulfonic acid (4.5 g) was added to a solution of 300 g ofepoxidized linseed oil in 1 l of dry benzene. The mixture was refluxedfor 5 hours. A solution of 5 g of sodium bicarbonate in 100 ml of waterwas added, then the benzene was removed at reduced pressure. The oilyresidue was then dissolved in 1 l of ethyl acetate (to reduce emulsionproblems), washed with 200 ml of water and dried over anhydrous sodiumsulfate. Removal of the solvent under reduced pressure yielded 245 g ofyellow oil. The triglyceride product, denoted Cpd. A, was evaluated inthe clinical study whose results are reported below in Example 6.

EXAMPLE 5 Saponification of Isomerization Product

The above triglyceride (245 g, 0.27 mol) was dissolved in 850 ml of 95%ethanol. A solution of 56 g (1.0 mol) of potassium hydroxide in 150 mlof water was added and the mixture refluxed for 2 hours. The ethanol wasremoved at reduced pressure, 1 l of water added, and the solutionextracted once with 350 ml of ethyl acetate to remove neutral compounds.The aqueous solution was acidified with 6N hydrochloric acid to pH 3 andextracted with three 250 ml portions of ethyl acetate. The combinedorganic phases were washed once with 250 ml of water and dried overanhydrous sodium sulfate. Removal of the solvent under reduced pressureyielded 204 g of pale yellow oil. The free acid form, denoted Cpd. B,was evaluated in the clinical study whose results are reported below inExample 6.

EXAMPLE 6 Clinical Study

The triglyceride product, denoted Cpd. A and described above in Example4, the free acid form, denoted Cpd. B and described above in Example 5,and a placebo consisting of raw linseed oil, were administered in aclinical study to fifteen children, aged 6 to 15 years, over a period of3 months. The fifteen patients used in this study were homozygous sicklecell patients who had had severe clinical courses. During the study thegeneral condition of the children, their clinical symptoms, hematology,and biochemistry were observed.

At the beginning of the study, each patient was hospitalized for 10days. A physical examination and laboratory tests were done to establishbaseline activity levels of various body systems, including a completeblood count, liver function tests for serum bilirubin, alkalinephosphatase, transaminases, and serum protein, serum electrolytes,creatinine, BUN (blood urea nitrogen), and uric acid. On the second day,the patients started taking the drug and continued doing so for 10 days.Each patient was carefully monitored to check for possible side effects.At the end of the 10 days, the physical examination and laboratory testswere repeated and the patients discharged.

Drug administration continued on an out-patient basis and the patientswere seen for evaluation once a week. At each visit, each patient wassupplied with a week's course of the drug and was questioned about anysubjective feeling which could indicate drug side effects, such asnausea, vomiting, diarrhea, and dizziness. The patient was also askedabout the occurrence of crises or other problems. A physical examinationwas carried out and laboratory tests were run again. The trial lasted 3months.

To facilitate administration, the compound was admixed with an inertmedium, 100 mg of the compound in 75 mg of mineral oil in a gel capsule.The fifteen patients were divided into 3 groups of 5 (3 males and 2females). The patients in group 1 received gel capsules of Cpd. A inmineral oil. The patients in group 2 received gel capsules of Cpd. B inmineral oil. The patients in group 3 received gel capsules of rawlinseed oil in mineral oil as a placebo. Each patient received 1 gelcapsule a day, to ensure good serum levels.

The fifteen patients used in this study were homozygous sickle cellpatients who had had severe clinical courses. They were all severelyretarded in physical growth, and had all been hospitalized at leastonce. They all had stigmata of sickle cell anemia, that is skullbossing, gnathopathy, and hepatomegaly.

The only other medications taken by the patients during the study werethe traditional folic acid and prophylactic anti-malarial medication.

Cpds. A and B and the placebo were tolerated well by the 15 patients.Not one patient complained of nausea, vomiting, dizziness, or diarrhea.A few comments were made concerning the taste of the medication.

At no time during the study was there any indication of bone marrowdepression in the patients treated with Cpds. A and B. The hematocritdid not fall significantly in any of the patients at any time during thestudy. The total white blood cell count and differential counts did notshow any adverse variation. The same applied to the platelet count.

There was no evidence, in the patients treated with Cpds. A and B, ofhepato-toxicity, expressed, either as cholestasis or hepatocellulardamage. In fact, there was a significant decrease in the combined meanvalue of the total serum bilirubin level before and after the trial. Theconjugated bilirubin level and the serum alkaline phosphataseconcentration also showed a sequential decrease in some of the patients.

Renal function was monitored by estimating the patients' serumcreatinine and BUN levels serially. These parameters remained withinnormal limits, with no appreciable increase in their values in all thepatients except for those in group 3. In fact when the BUN values forthe patients treated with Cpds. A and B were considered together, therewas a statistically significant decrease in the mean value at the end ofthe trial, in comparison to the initial value. The serum electrolytesand serum uric acid values remained normal, in the 10 patients in groups1 and 2 throughout the duration of the study.

One of the established complications of sickle cell anemia isretardation of physical growth. The 10 patients in groups 1 and 2 ofthis study had marked stunting of physical growth. Each of them weighedbelow 55% of the expected weight for his/her age. In normal children andadolescents, the age of the patients in this study, the expectedincrease in weight per year is about 2.8 kg. This figure is, of course,much lower in sickle cell patients. It is, therefore noteworthy thateach of the 10 patients gained considerable weight, ranging from 0.9 to2.5 kg, in the 3 months, or less, that they were being treated with CpdsA or B. The mean weight at the end of the study was statistically higherthan the initial value. Although anthropometric data were not alwaysdocumented on routine clinical visits before the study, in the 10patients for whom there was such documentation, none had shown thisdegree of weight gain before.

Hepatic enlargement is a common complication of sickle cell anemia andit has been documented in 30 to 50% of children with the disease. Thecause of this, usually, is the blockage of the hepatic sinusoids bysickled erythrocytes, with consequent congestion.

The 15 patients in this study all had hepatomegaly, ranging from 5 to 13cm (mean 6.9 cm) at the onset of the trial. Within 10 days of startingtreatment with Cpds. A and B, there was noticeable reduction in all 10patients in groups 1 and 2. By the end of the study, the liver was nolonger palpable in one and was about 1 cm in another. The mean of thepalpable liver size was 2.9 cm for the patients in groups 1 and 2, atthe end of the study. The difference between this value and the initialone was highly significant.

This reduction in liver enlargement was the most striking uniformfinding in these patients during this study. Three of the patients hadhad persistent enlargement of the liver for as long as they had beenattending the sickle cell clinic before the study. It is, therefore,highly improbable that this uniform reduction in liver enlargement was achance finding.

The other evidence for a beneficial effect of Cpds. A and B on hepaticfunction is the significant reduction in the mean value of the totalserum bilirubin at the end of the trial. It is also noteworthy that theserum conjugated bilirubin fell sequentially in the 5 patients that hadappreciably elevated values, at the beginning of the study. When thesefindings are considered along with the reduction in hepatic size, itcould be inferred that Cpds. A and B reduced the degree of sinusoidalblockage (and obstructive jaundice) because of its antisicklingproperties.

The fact that Cpds. A and B possess anti-sickling properties isreflected by a reduction in the frequency of severe pain crises. All thepatients in this study had frequent pain crises before they startedtaking the medication tested in this study. However, during the study,there was not a single episode of pain crisis that necessitatedanalgesic administration, in the patients who were treated with Cpds. Aand B.

Cpds. A and B were shown in this study to be well accepted, non-toxicand may, indeed, be useful in the management of sickle cell disease.During this study, these drugs have demonstrated positive effects onbody weight, hepatic function, and the frequency of severe pain crisis.

What is claimed is:
 1. A method of making a compound for the treatmentof sickle cell disease which comprisesthe partial epoxidation of acompound which is a member of the group consisting of methyl linolenateand linseed oil, said partial epoxidation resulting in epoxide products;and the isomerization of said epoxide products using p-toluenesulfonicacid.
 2. The method of claim 1, wherein said compound is linseed oil. 3.The method of claim 2, wherein after said isomerization step, theproduct of said isomerization step is saponified.